Image capture for large analyte arrays

ABSTRACT

Analyte arrays such as solutes in a slab-shaped gel following electrophoresis, and particularly arrays that are in excess of 3 cm square and up to 25 cm square and higher, are imaged at distances of 5 cm or less by either forming sub-images of the entire array and stitching together the sub-images by computer-based stitching technology, or by using an array of thin-film photoresponsive elements that is coextensive with the analyte array to form a single image of the array.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/056,162, filed Oct. 17, 2013, which claims priority to U.S.Provisional Patent Application No. 61/715,103, entitled “Image Capturefor Large Analyte Arrays” and filed Oct. 17, 2012, as well as to U.S.Provisional Patent Application No. 61/815,456, entitled “Image Capturefor Large Analyte Arrays” and filed Apr. 24, 2013. Each priorityapplication is herein incorporated by reference in its entirety for allpurposes.

BACKGROUND OF THE INVENTION

Many procedures that are performed in biochemical laboratories involveanalyses of multiple samples or materials distributed over atwo-dimensional area. Examples of such procedures are screening studiesperformed on substances that are placed in individual wells of amulti-well plate such as a standard 96-well microtiter plate or largerplates, or on molecular species that are applied as droplets orregularly spaced spots, either microscopic in size or larger, on a solidsurface. Further examples are slab-shaped electrophoresis gels in whicheither two-dimensional electrophoretic separations or one-dimensionalseparations of multiple samples in parallel have been performed. Stillfurther examples are blotting membranes to which electrophoreticallyseparated species in the form of spots or bands have been transferredfrom a slab gel. Other examples will readily occur to the skilledbiochemist. In all of these examples, detections and analyses of theindividual sites in the two-dimensional array are often achieved bylight energy associated with each site, and may consist simply ofdeterminations of the presence or absence of particular species or mayalso include quantitative determinations, either on an absolute basis oras comparisons among different sites. The light energy can betransmissive, absorptive, reflective, or generated by the materials atthe sites themselves. Species in electrophoresis gels or blottingmembranes, for example, are commonly detected by fluorescence,chemiluminescence, or bioluminescence, either as inherentcharacteristics of the species at the sites or as a result of treatmentof the species once they are separated throughout the two-dimensionalarray. The treatment may include binding reactions in whichenergy-emitting labels are attached to the species, or irradiation ofthe species or the labels with excitation energy that will cause them toemit light energy, most often at different wavelengths.

The two-dimensional array, or the planar matrix supporting the array,can be relatively large, for example exceeding about 5 cm, 10 cm, 20 cm,or even 30 cm, in either length, width, or both. In terms of ranges, thelength, width, or both, can for example be from about 3 cm to about 100cm, from about 10 cm to about 75 cm, from about 5 cm to about 50 cm, orfrom about 5 cm to about 25 cm. In some cases, a large two-dimensionalarray is divided into a number of narrow strips for independentanalysis. In these cases, one may wish to analyze a single strip, two ormore but less than all strips, or all strips of the array. When any ofthese images is taken by a camera, including digital cameras, the arraysare large enough to require placing the camera a considerable distancefrom the array. One consequence of placing the camera far from the arrayis that less light is captured. More light can be captured by placingthe camera or image acquisition device closer to the array, but thisoften requires the use of multiple lenses and other optical componentsto obtain the full image. Some lenses distort the image and geometricresolution can be of limited quality. Intensity roll-off furtherdistorts the image. A type of camera that is commonly used for imagingin biochemical applications is a charge coupled device (CCD) camera, buteven with such a camera and its lenses and other optical components, thesensors must be placed well above the object plane, causing the entireapparatus to consume a large amount of space in the laboratory.

SUMMARY OF THE INVENTION

It has now been discovered that analytes that are distributed in aplanar array whose lateral dimensions are too large to be imaged withoutdistortion by a camera that is less than approximately 5 cm from thearray can indeed be accurately imaged at a distance of 5 cm or less.This can be achieved by forming sub-images of segments of the analytearray and using image stitching technology to combine the sub-imagesinto an image of the entire analyte array. Alternatively, a single imageof the entire analyte array can be obtained by use of a thin-film arrayof photoresponsive elements. The object to be imaged can be the analytearray itself or the planar area in which the analyte array resides andwhich typically extends beyond the extremities of the analyte array. Theplanar area can be a planar support matrix for the analyte array, andthe matrix can be a multi-well plate, a gel, a blotting membrane, or anysurface on which the array has been distributed. When sub-images ofsegments of the analyte array or supporting planar matrix are used, theycan be formed by solid-state image sensors arranged in a two-dimensionalarray. One or more of the image sensors can be moved or repositionedwith respect to the support matrix to acquire multiple sub-images. Whena single image is to be formed, this can be achieved by a thin-filmarray of photoresponsive elements that is coextensive with the analytearray or planar matrix, or larger, combined with thin-film addressingand signal processing circuitry that accesses each of thephotoresponsive elements and directs the energy accumulated by eachelement to an image storage or display device where it is stored ordisplayed in accordance with the location of each element relative tothe array to form the image. A transparent faceplate, such as a fiberoptic faceplate or fiber optic taper, can be placed between the imagesensors or thin-film array and the analyte array. The transparentfaceplate can provide mechanical support to the analyte array andprotect the image sensors or thin-film array from damage, such as canoccur when the analyte array is wet.

Provided herein is a method of analyzing a plurality of analytesdetectable by light emission and arranged in a two-dimensional array.The array is supported by a planar matrix whose length, width, or bothlength and width measure a minimum of about 3 cm. The method includesplacing the planar matrix within 5 cm of a detector. The detector can beeither: (1) a plurality of solid-state image sensors that are arrangedin a sensor array and that are each positioned to form a sub-image of asegment of the matrix such that the segments collectively cover theentire matrix, and a computer for assembling the sub-images formed ateach of the image sensors in accordance with the positions of the imagesensors in the sensor array to form an image of the planar matrix infull as a composite of the sub-images; or (2) a plurality ofphotoresponsive elements arranged in an array that is at leastsubstantially coextensive with the matrix, thin-film addressingcircuitry that controls accumulation of energy by, and release of energyfrom, the photoresponsive elements, and a data storage medium thatcorrelates energy released from the photoresponsive elements with siteson the planar matrix and forms an image of the planar matrix in fullfrom the energy so released.

The method also includes generating the planar matrix image in full bythe detector, in a manner that either compensates for or eliminates anyirregularities in light intensity across the planar matrix image thatare not representative of the two-dimensional array of analytes; andanalyzing the analytes from the planar matrix image so generated.

In some embodiments of the method, generating the planar matrix image infull includes applying flat field correction to compensate for oreliminate the irregularities.

In some embodiments of the method, the planar matrix is a slab-shapedgel and the two-dimensional array is generated by electrophoreticseparation of the analytes within the gel. In other embodiments, theplanar matrix is a blotting membrane and the two-dimensional array is anarray of solute bands transferred to the blotting membrane from aslab-shaped gel in which the bands were generated by electrophoreticseparation of the analytes within the gel.

In some embodiments of the method, the detector includes a plurality ofsolid-state image sensors that are arranged in a sensor array and thatare each positioned to form a sub-image of a segment of the matrix suchthat the segments collectively cover the entire matrix, and a computerfor assembling the sub-images formed at each of the image sensors inaccordance with the positions of the image sensors in the sensor arrayto form the image of the planar matrix in full as a composite of thesub-images. In one such embodiment, the solid-state image sensors areCCD or CMOS sensors, and the computer includes computer-readableinstructions for registering the sub-images, for calibrating thesub-images, and for merging overlapping regions between neighboringsub-images.

In other embodiments of the method, the detector includes a plurality ofphotoresponsive elements arranged in an array that is at leastsubstantially coextensive with the matrix, thin-film addressingcircuitry that controls accumulation of energy by, and release of energyfrom, the photoresponsive elements, and a data storage medium thatcorrelates energy released from the photoresponsive elements with siteson the planar matrix and forms the image of the planar matrix in fullfrom the energy so released. In one such embodiment, the photoresponsiveelements are photodiodes and the thin-firm addressing circuitry includesthin-film field effect transistors.

In some embodiments, the detector defines a planar detection surface,and the method further includes generating a dark signal pattern of eachsub-image and subtracting the dark signal pattern from the planar matriximage prior to analyzing the analytes. In some embodiments, the detectordefines a planar detection surface, and the method further includesmeasuring temperature at selected sites along the planar detectionsurface to determine a temperature pattern, generating a dark signalpattern representative of the temperature pattern, and subtracting thedark signal pattern from the planar matrix image.

In some embodiments of the method, the detector is comprised of pixelsand the planar matrix image is generated by passing light from theplanar matrix through an array of light pipes to the detector, such thateach light pipe directs light to a single pixel.

In some embodiments of the method, a transparent faceplate is placedbetween the planar matrix and the detector. The transparent faceplatecan be, for example, a fiber faceplate or a fiber taper. In some suchembodiments, the maximum thickness of the transparent faceplate is about0.1, 1, 2, 5, 10, 20, or 50 mm.

An additional method is also provided for analyzing a plurality ofanalytes detectable by light emission and arranged in a two-dimensionalanalyte array supported by a planar matrix. This method includes:placing the planar matrix within 5 cm of a detector comprising one ormore moveable solid-state image sensors; moving the image sensor(s) withrespect to the planar matrix and acquiring a plurality of sub-images ofthe planar matrix; assembling the sub-images into a full image of theplanar matrix in accordance with the positions occupied by the imagesensor(s) when the sub-images are acquired; and analyzing the analytesusing the full image of the planar matrix.

In some embodiments of the additional method, the length, width, or bothlength and width of the planar matrix measure a minimum of about 3 cm.In some embodiments, assembling includes stitching together at least twoof the sub-images. In some embodiments, assembling includes juxtaposingsub-images acquired by the same image sensor.

The additional method can also include the step of compensating for oreliminating any irregularities in light intensity across the full imageof the planar matrix, where the irregularities are not representative ofthe two-dimensional analyte array.

In some embodiments of the additional method, a transparent faceplate isplaced between the planar matrix and the detector. The transparentfaceplate can be, for example, a fiber faceplate or a fiber taper. Thetransparent faceplate can provide mechanical support to the analytearray. In some such embodiments, the maximum thickness of thetransparent faceplate is about 0.1, 1, 2, 5, 10, 20, or 50 mm.

Details of these and other features of the invention are presented inthe sections that follow.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1A shows flat field correction of a single image or sub-image of areference plate. FIG. 1B shows flat field correction and normalizationof adjacent sub-images of a reference plate or analyte array. Thenumbers in bold correspond to pixels that overlap between the twosub-images. Overlap results from the image sensor(s) used to acquire thesub-images being focused on the same area of the reference plate oranalyte array. Flat field correction is first applied to each sub-imageindividually. Then the pixel values for one sub-image are normalized tothose of the other sub-image according to the values of the overlappingpixels in the two sub-images.

FIG. 2 shows mechanical interference or contact between an image sensorand a sample during scanning of the sensor over the sample. Suchinterference is undesirable and can be avoided in some embodiments ofthe methods and apparatus presented herein.

FIG. 3 shows analyzing of a plurality of analytes arranged in atwo-dimensional analyte array supported by a planar matrix with atransparent faceplate placed between the planar matrix and the detector.

FIG. 4 shows analyzing of a plurality of analytes arranged in atwo-dimensional analyte array supported by a planar matrix with atransparent faceplate formed as a fiber optic taper placed between theplanar matrix and the detector.

DESCRIPTION OF SELECTED EMBODIMENTS

In embodiments involving the use of a plurality of solid-state imagesensors forming images of sub-areas of the matrix, such images referredto herein as “sub-images,” and stitching together the sub-images in amosaic to form a full image of the matrix can be achieved by imagestitching technologies of the prior art. Examples of image stitchingtechnologies are those that include algorithms for aligning orregistering the sub-images by mapping the coordinates of each sub-imageto assure that the sub-images are assembled in the correct geometriclocations relative to each other, and algorithms for eliminating visibleseams by removing or merging overlapping regions between neighboringsub-images. Additional algorithms can also be included in certainembodiments, such as algorithms for calibration of the sub-images andfor ghost removal.

Each sensor can contain a two-dimensional array of pixels to produce itssub-image, and the array can be rectilinear. The number of pixels willbe great enough to produce an image of the sub-area that shows at leastapproximate outlines of the shapes of any spots or bands in the area,but in general the number of pixels can vary. In some examples, thenumber of pixels in the pixel array ranges from about 5×5 (5 rows and 5columns) to about 1,000×1,000 (1,000 rows and 1,000 columns). Analternate range is from about 10×10 to about 100×100. Each image sensorcan be a digital camera whose sensors are either charge-coupled device(CCD) sensors or complementary metal-oxide-semiconductor (CMOS) sensors,or other arrays of photosensitive elements. Beneficial results can beobtained by equipping each digital camera with a lens, selected suchthat all lenses for all cameras have the same focal length, the focalplanes of all cameras are coplanar, and the optical axes of all camerasare parallel to each other.

In some embodiments, the number of sub-images that are stitched togetherinto a full image is equal to the number of image sensors. In theseembodiments, the image sensors collectively image an area approximatelyequal to, or greater than, the area of the entire matrix or analytearray. In other embodiments, by contrast, some image sensors acquiremultiple sub-images. These sensors, herein called “moveable sensors”,can be repositioned with respect to the matrix, with a new sub-imagebeing acquired at each position, in order for the plurality of imagesensors to image the entire matrix.

A moveable sensor can be moved or repositioned as desired. For example,the sensor can be swept in a line or raster-scanned over an area. Thiscan be accomplished by attaching the sensor to a mechanical positionerof the kind used in document scanners or photocopiers. Alternatively,the matrix can be moved while the sensor is held fixed. The portions ofthe matrix corresponding to the sub-images acquired by one moveablesensor can likewise be arranged in any pattern, such as in a line orgrid. In some embodiments, the pixel array contained in a moveablesensor can have equal numbers of rows and columns, as discussed above.In other embodiments, the number of rows and columns in a pixel arraycan be unequal, with the result that a sub-image is made up of arectangular or linear array of pixels rather than a square array. If thepixel array is aligned with the direction of movement of the moveablesensor, then having fewer pixels along the direction of movement meansthat smaller movements are needed to capture consecutive sub-images.

Movement of the sensor with respect to the matrix can occur in discretesteps, with each step corresponding to a distinct sub-image, or can becontinuous, with sub-images being acquired at regular time intervalsappropriate for the rate of movement. In the latter case, theacquisition time should be short compared with the time interval, sothat effectively no movement occurs during acquisition. Stitching ofsub-images acquired by a moveable sensor can be performed as discussedabove, such as by aligning any overlapping elements in the sub-images,or simply by juxtaposing sub-images resulting from consecutiveacquisitions. The latter option reduces the amount of image processingthat must be performed to obtain the full image. In some embodiments,the matrix can be imaged using a combination of fixed and moveable imagesensors. In other embodiments, only moveable image sensors are used. Forexample, several image sensors can be arranged in a line, and can beswept over the matrix in concert to obtain a full image of the matrix.

The composite image can be evaluated qualitatively or quantitatively.Quantitative analysis can be used to determine the composition of thesample on which the electrophoresis was performed in terms of thepresence or absence of known molecular species in the sample. Suchanalysis can include correlating each band or spot on the image with asingle molecular species or an electrophoretically co-migrating group ofspecies. The correlation can be achieved by comparing thetwo-dimensional coordinates of the band or spot with those of a standardtemplate in which all positions are identified by their coordinates andby the species that reside at those coordinates. Alternatively, thecorrelation can be achieved by including standards in theelectrophoresis gel in which electrophoretic separation has taken place,alongside or together with the sample or samples to be analyzed. Thestandards can be mixtures of the same molecular species suspected to beincluded, or potentially included, in the sample(s), or a series ofmolecular weight markers spanning the range of molecular weights thatare typical among molecular species in samples having the same or asimilar origin as the sample(s) being analyzed. The correlation can beperformed visually by a laboratory technician, or it can be performedelectronically. Visual correlations can be achieved by projecting thecomposite image onto photographic media or a computer screen. Electroniccorrelations can be achieved by computer software that collects theinformation from the pixels and sorts the collected informationaccording to the pixel location, while comparing the information to alibrary of such information or to standards as described above toidentify each spot or band as representing a particular molecularspecies.

When quantitative analysis is desired, sensors can be used that collectdata over a period of time such that each pixel will have an amount ofretained data proportional to the intensity of the emission from thesite on the matrix that the pixel is focused on. An electronic signalproportional to the accumulated data for each pixel can then beprocessed and compared to corresponding signals from other pixels todetermine relative intensities and thereby the relative amounts of themolecular species. Absolute values can be determined by calibration orby comparison to standards representing known concentrations or amountsof the species. All such signal processing and comparisons can be doneby computer, using well-known algorithms, which detect and record thelocation of each pixel and of the location within the matrix that eachpixel represents, and the presence or absence of a signal as well as thesignal intensity.

Image generation in the practice of this invention is performed, whennecessary, in a manner that eliminates or compensates for anyirregularities in light intensity across the planar matrix image thatare not attributable to or representative of the analyte array. Theexpression “across the planar matrix image” is used herein to refer todirections along the surface of the image, i.e., within the plane of theimage. These irregularities tend to introduce artifacts, shadings, orlight intensity variations in general that do not represent differencesamong the analytes, and they can arise in various ways. Individualcameras, for example, inherently produce distorted images arising fromirregularities among the pixels that form the images. Such distortion isoften due to nonuniformities in the optical system, or to thepositioning system when moveable sensors are employed, but can arisefrom other artifacts as well, and in any case interferes with accuratequantitation between the pixels in the array. Compensation for thisdistortion can be achieved by a calibration technique known in the artas “flat field correction.” Examples of flat field correction can befound in Naghieh, H. R., et al., United States Patent ApplicationPublication No. US 2003-0039383 A1 (published Feb. 27, 2003), and inU.S. Pat. No. 5,799,773 (issued Sep. 1, 1998),U.S. Pat. No. 5,891,314(issued Apr. 6, 1999), U.S. Pat. No. 5,897,760 (issued Apr. 27, 1999),and U.S. Pat. No. 5,951,838 (issued Sep. 14, 1999) (all listingHeffelfinger, D. M., and C. Van Horn as inventors). The methodsdescribed in the Heffelfinger and Van Horn patents variously includecalibrations of the lens and detector assemblies, use of a scanninglight source to achieve uniform illumination, use of a mirror orbeamsplitter to sample the source, or the generation of correction dataover a range of aperture and magnification settings. The methoddescribed in the Naghieh et al. publication involves comparing the imageof the array of interest to the image of a reference plate that respondsto incident light uniformly along its length and width. Thus, forexample, the reference plate is uniformly absorptive and/or transmissiveof light, or contains fluorescent material uniformly distributedthroughout the plate and is uniformly excitable by incident light. Thereference plate is placed in the imaging system independently of, and inplace of, the array sought to be imaged, and an image of the referenceplate is taken in the same manner as the image of the array. The twoimages are then compared on a pixel-by-pixel basis, and the gel image iscorrected by an appropriate formula or algorithm that accounts for anynon-uniformities or deviations in the reference plate image. The imageof the reference plate is termed a flat field image, and the correctedimage of the gel is termed a flat field-corrected image.

The flat-field correction technique of Naghieh et al. will be describedin more detail, with the understanding however that this is but oneexample of such a technique.

In accordance with the Naghieh et al. technique, the reference plate canbe a flat plate having the same dimensions as the array sought to beimaged or, when only a portion of the array is of interest thedimensions of the reference plate will be at least as great as those ofthe portion. The plate can be from one-sixteenth inch (0.16 cm) toone-half inch (1.27 cm) in thickness, or as an example, approximatelyone-eighth inch (0.32 cm) in thickness. The plate will be one thatresponds to incident light uniformly along its length and width, i.e.,it contains no nonuniformities that would cause it to either absorb ortransmit light differently at any point on the plate than at any otherpoint. When the plate is a fluorescent plate, it will be one thattransmits light without transmitting an image of the light source andwill either be colored with a fluorescent dye or white. The plate willbe constructed to disperse the light striking it from the light sourceand to emit the light toward the detector in a manner that includes nospatial variations other than those attributable to the light source.For arrays that contain fluorescent labels, a particularly usefulreference plate is one that has a fluorescent dye, such as a red ororange dye. For arrays that involve absorption of light from the lightsource rather than emission, a translucent fluorescent white referenceplate that converts ultraviolet light from the light source to whitelight is particularly useful.

The image of the analyte array can be taken either before or after theimage of the reference plate. In either case, the image of the referenceplate can be stored as digital data for use in correcting the image ofthe analyte array. Correction can be achieved by any formula oralgorithm that compares the two images and corrects the analyte arrayimage on the basis of nonuniformities or deviations in the referenceimage (FIG. 1A). This comparison and correction are readily performed bysoftware, which can then display the corrected image. When the imagesconsist of two-dimensional arrays of pixels whose locations in the arrayare defined by orthogonal coordinates X and Y, one example of correctionformula is as follows:

${{Piff}({XY})} = {{{Pi}({XY})} \times \left( \frac{{Av}_{Flat}}{{P({XY})}_{Flat}} \right)}$in which:

Piff(XY) is the corrected value of the pixel at position XY

Pi(XY) is the value of the pixel at position XY before correction

AV _(Flat) is a coefficient obtained from the average of the valuesobtained with the reference plate, and

P(XV)_(Flat) is the value of the pixel at position XY of the referenceplate.

Other algorithms and methods of correction will be readily apparent tothose skilled in the art. Once the correction has been made, thecorrected pixels can be reassembled to form the corrected image.

In embodiments of the invention in which imaging of the analyte array isachieved by first forming sub-images or segments of the analyte arraywith individual image sensors or cameras, flat field correction can beperformed on the composite image to compensate for differences inresponse between the various cameras, or differences for the same cameraarising over time or at various locations. Using adjacent sub-images asan example, this type of correction can be achieved by focusing facingedges of the two sub-images on the same line of pixels, thereby causingthe two sub-images to overlap at these pixels. A comparison between theintensities of these pixels in one sub-image with those of the othersub-image will provide a correction factor, which can then be applied toall pixels of one sub-image, thereby normalizing the two sub-images(FIG. 1B). Flat field correction can thus be used both within eachsub-image and between different sub-images.

Descriptions of the formation of sub-images and the stitching togetherof sub-images to achieve a composite image are found in Shimizu (CanonKabushiki Kaisha) U.S. Pat. No. 4,675,533 (Jun. 23, 1987), Gruber et al.(Vexcel Imaging GmbH) U.S. Pat. No. 7,009,638 B2 (Mar. 7, 2006),Ziemkowski (Hewlett-Packard Development Company) U.S. Pat. No. 7,136,094(Nov. 14, 2006), Agarwala et al. (Microsoft Corporation) U.S. Pat. No.7,499,586 B2 (Mar. 3, 2009), Ojanen et al. (Nokia Corporation) U.S. Pat.No. 7,499,600 B2 (Mar. 3, 2009), and Regents of the University ofMinnesota International Patent Application Publication No. WO 02/093144A1 (Nov. 21, 2002).

In embodiments of the invention that utilize an array of photoresponsiveelements, the lateral dimensions of the array are the same size as thematrix or larger, i.e., the array is at least coextensive with thematrix, and a single image of the entire matrix that is at leastapproximately the same size as the matrix is formed on the array. Thenumber of photoresponsive elements in the array can vary widely, butwill most often be at least 1,000×1,000, and in many cases within therange of 1,000×1,000 to 1,000,000×1,000,000, or even a range of1,000×1,000 to 10,000,000×10,000,000. The photoresponsive elementsreceive light energy from the sites on the matrix with which theelements are aligned, and the elements generate detectable electricalsignals that are representative of the energy that they receive from thematrix. The electrical signals are stored in a data storage medium fromwhich they can be projected or otherwise made visible or detectable asan image of the matrix.

Any of a variety of known photoresponsive elements can be used, examplesof which are photodiodes, phototransistors, photoresistors, andphotovoltaic devices. The photoresponsive elements include at least onelayer of semiconductor material, preferably silicon semiconductor alloymaterial, examples of which are amorphous silicon alloy materials,amorphous germanium alloy materials, amorphous silicon carbon alloymaterials, and amorphous silicon germanium alloy materials. Whenphotodiodes are used, each one can include two oppositely doped layersof the semiconductor material, optionally with a layer of intrinsicsemiconductor material interposed between them, thereby forming a p-i-ntype photodiode. In certain embodiments, the array further includes anaddressing mechanism for independently accessing each photoresponsiveelement. An example of such a mechanism is one that includeselectrically conductive lines arranged in an x-y matrix and a blockingelement associated with each photoresponsive element, such as a diode, atransistor, a resistor, a threshold switch, or a relay. The addressingcircuitry can either be separate from the sensor array or integratedwith the sensor array on a common substrate.

Photosensors and their associated components for this type of array areknown in the art. Examples are certain sensors developed for x-rayimaging, particularly those made for large areas and that involve lowread noise and low dark current noise. Two technologies for x-rayimaging that utilize sensors of this type are thin-film field effecttransistor (TFT) technology and CMOS. The pixel elements used in thearrays in these technologies are generally large (50-200 μm), a featurethat makes these arrays particularly useful in the practice of thepresent invention. Thus, in certain embodiments, x-ray imagingtechniques are used for gel documentation and chemiluminescence imaging.

Architectures vary widely among different photosensors, and a generalunderstanding of the fundamental structure and function of photosensorsfor use in this invention can be obtained by consideration of oneillustrative class of photosensors, i.e., that of a combination of aphotodiode and a TFT. The architectures and fabrication methods of TFTsvary widely as well, but according to one example, a 1200 Å layer oftitanium-tungsten (TiW), chromium, molybdenum, or tantalum, is firstformed over an 800 Å layer of aluminum on a substrate to serve as ametal gate electrode. A 3000 Å gate dielectric layer of silicon nitride(SiN_(x)) is then formed over the metal gate electrode, and a 300-500 Ålayer of hydrogenated amorphous silicon (a-Si:H) is formed over the gatedielectric layer. A 1500 Å etch stopper layer of SiN_(x) is formed overthe a-Si:H layer above the gate electrode, and a 500-1000 Å n+ layer isformed over the a-Si:H layer and partially over the etch stopper layer.A 500 Å TiW layer is formed over the n+ layer and a 0.5μ Al layer isformed over the TiW layer to serve as a barrier preventing the Al layerfrom interacting with the n+ layer. The n+ layer, the TiW metal layer,and the Al layer on the left side of the etch stopper serve as thesource electrode of the TFT, and the n+ layer, the TiW metal layer andthe Al layer on the right side of the etch stopper serve as the drainelectrode of the TFT. A 0.5-2.0μ silicon oxynitride (SiON) layer with avia hole is formed over the TFT, and a 500-1000 Å n+ doped layer isformed over the SiON layer, making contact with the drain electrode. Anundoped 0.5-2.0μ a-Si:H layer is formed over the n+ doped layer, a 100 Åp+ doped layer is formed over the undoped a-Si:H layer, and a 500-1000 Åindium-tin-oxide (ITO) transparent conductive layer is formed over thep+ doped layer. A 0.5-2.0μ silicon oxynitride (SiON) layer with a viahole is formed over the conductive layer, and a bias contact, which is a500 Å layer of TiW beneath a 0.5-1.0μ layer of Al, is formed over theSiON layer, contacting the conductive layer. Finally, a 0.5-1.0μpassivation layer of SiON is formed over both the conductive layer andthe bias contact. Adjacent photodiodes are separated by a notch passingthrough the n+ doped layer, the a-Si:H layer, the p+ doped layer.

The photodiode can be formed over the TFT in a variety of ways.According to one example, a SiON layer is formed over the TFT, thenmasked and etched to form a via hole exposing the drain electrode. An n+doped layer is then formed over the SiON layer making contact with thedrain electrode. An a-Si:H layer is then formed over the n+ doped layer,a p+ doped layer is formed over the a-Si:H layer, and a conductive layeris formed over the p+ doped layer. The conductive layer is masked andetched to form a notch that exposes the SiON layer. A SiON layer is thenformed over the conductive layer and fills the notch, thereby preventingthe metal bias layer from shorting out the photodiode and from providinga connection across the etch through the n+ layer. The SiON layer isthen masked and etched to form a via hole, and a metal bias layer isformed over the SiON layer to contact the conductive layer through thevia hole. The metal bias layer is then masked and etched to form a biascontact. A passivation layer is then formed over both the conductivelayer and the bias contact to complete the photodiode. The fillednotches thus isolate the photodiodes from each other so that they canaccumulate charges independently. Each photodiode is biased by applyinga voltage on the bias contact which induces an electric field in thea-Si:H layer. When light enters the a-Si:H layer, electron-hole pairsare generated and are swept by the electric field to opposite sides ofthe photodiode where they accumulate near the conductive layer and then+ doped layer. During operation, the TFTs are turned OFF to allow thephotodiodes to accumulate charge based on incident light.

Upon receipt of a control signal from an external controller, a TFTturns ON and, the accumulated charge is allowed to flow as currentthrough source electrode to components that amplify and process thereceived image signal.

The electrical signals generated by the photosensor array can beconveyed to a conventional image storage or display device. Examples aremagnetic, optical, semiconductor and bubble memory devices. A videodisplay terminal, a photographic film, or a phase change optical datastorage medium can be used. The camera that contains the photosensorarray can contain a trigger mechanism for initiating the reading of theinformation that is released by the TFTs and a mechanism for writing theinformation thus read onto the storage medium. The resulting image canbe analyzed visually on a qualitative or quantitative basis, either by alaboratory technician or by instrumentation.

Within each image or sub-image, an amount of dark signal of a magnitudethat is great enough to impair or limit the sensitivity of the sensorcan occur. “Dark signal” is defined as the response of a photosensitiveelement in the absence of light. One source of sensor limitation fromdark signal is fundamental shot noise. Another is fixed pattern noise.For large area sensors, noise from dark signal can become very large dueto the large size of the sensor array. Physical processes that producedark signal usually increase with increasing amounts of material used inthe photosensitive element. The sensitivity of the photosensor array canbe increased significantly by removing fixed pattern dark noise. Thiscan be achieved by producing a dark signal pattern for the sensor arrayand subtracting the dark signal pattern from the measurement patterngenerated by the array. The dark signal pattern can vary withtemperature, time, or both, and can thus be characterized as a functionof these two variables. For example, a temperature measurement can bemade and the dark current pattern can be adjusted accordingly tosubtract the fixed pattern dark current. The temperature can also varybetween different photosensors within the photosensor array. Tocompensate for this, a plurality of temperature measurements can betaken at different locations within the photosensor array, i.e., atselected sites along the planar detection surface formed by the array,to determine temperature variations along the surface and the resultingtemperature pattern. The dark signal pattern can then be adjustedaccordingly.

Image sensors at times experience anomalous events that cause individualpixels or groups of pixels to register an abnormal responses whencompared to adjacent pixels, responses that are not representative ofthe sample. Such events are caused, for example, by externalelectromagnetic radiation or impacts of high energy particles from spaceor radioactive material, in addition to the dark signal cited above,while the origins of certain of the responses are not understood. Thefrequency of these anomalous events increases with increasing area ofthe photosensor array. The responses of the pixels to these events canbe corrected by various signal processing techniques. For example, onecan determine whether a particular pixel is undergoing an anomalousresponse by comparing the response at that pixel to those of neighboringpixels. If the response at the pixel is deemed not to be representativeof the measurement and thereby anomalous, the response at that pixel canbe corrected or removed.

Photosensor arrays often contain manufacturing defects or dark signaldefects introduced at the photosensor manufacturing stage, and suchdefects can have impacts on individual pixels, rows of pixels, columnsof pixels, or other sections of the sensor array. The number of defectstends to increase with increasing size of the photosensor array.Correction of these defects can improve both sensitivity and thequalitative appearance of image, as well as manufacturing yields, andsuch correction can be achieved by hardware, software, or both. Forexample, if an entire column of a photosensor array fails to functionproperly, that column can be replaced by simply averaging adjacentcolumns on either side.

Since the sample to be imaged (i.e., the analyte array) is placed inproximity to the photosensor array, one may wish to protect thephotosensor array from the sample, particularly with wet samples and/orsamples with which physical contact is to be avoided. Protection can beachieved by depositing or bonding a thin layer of material directly onor to the sensor surface to serve as a faceplate. The material must betransparent to the light being detected. A thin glass element with athickness approximately equal to or less than less than the size of apixel on the photosensor can be used as a faceplate, to avoidsignificant spreading of the light as it passes from the sample to thedetector surface. For example, a glass layer 100 micron in thickness canbe used, and the sample can be placed directly onto the glass layer andanalyzed without significant loss in spatial resolution. Any lightspreading and consequent loss in spatial resolution introduced by theprotective layer can be corrected by appropriate software algorithms.

Another means of improving the sensitivity of the photosensor array isto place a layer of material over the array that guides the light toeach sensor and thereby reduces or eliminates lateral diffusion toadjacent sensors to improve spatial resolution. An example of such amaterial is one that contains light pipes arranged in a pixelatedpattern, i.e., each pixel receiving light through a separate light pipe,or a separate group of light pipes. This can be achieved with a fiberoptic faceplate (also called a fiber faceplate), which is a coherentbundle of short optical fibers.

A transparent faceplate, for example a thin layer of glass or plastic ora fiber faceplate, can also be used in embodiments of the inventionwhere the sample is imaged using a plurality of solid-state imagesensors, and in particular where one or more sensors is scanned over thesample, as discussed above. Here, the faceplate can be placed betweenthe sample and the sensors, and can provide mechanical support to thesample. The faceplate also provides a flat surface that prevents contactbetween a sensor and the sample, which may occur if the sensor isscanned directly over the sample or if the sample is irregularly shaped(for example, due to being wet) (see FIGS. 2 and 3). Thus, the faceplateprevents damage to the sensor resulting from such contact, while thesample and sensor(s) remain close together. In embodiments where thetransparent faceplate provides mechanical support to the sample andprotects the sensor, the transparent faceplate is preferably a fiberfaceplate or made of a light-guiding material. This allows the faceplateto be thick enough to provide support without causing a loss ofresolution or sensitivity. A thick faceplate can be used when one ormore sensors are scanned over the sample, or in other embodiments wherethe sensors do not support the sample. By contrast, when the sample isdetected using a thin-film array, a thinner faceplate can be usedbecause the array mechanically supports the sample.

In embodiments of the invention using glass faceplates, a tradeoff isinvolved between supporting the sample and protecting the sensor on onehand, and allowing the high-resolution transmission of light betweenthem on the other hand. This is because, in this context, glass andother non-light-guiding materials offer greater strength but poorerresolution as the thickness of the faceplate is increased. Inembodiments using fiber faceplates, however, the faceplate can be madethick enough (e.g. 1-30 mm) to provide robust mechanical strength whilealso allowing transmission of light with very little loss or spreading.The fiber faceplate can be positioned so that the optical fibers makingup the faceplate are oriented parallel to the direction of lighttransmission. Fiber faceplates suitable for these embodiments areavailable from InCom (part no. B7D61-6) and Edmund Optics (part no.55-142), among others. In general, the maximum thickness of thetransparent faceplate can be about 0.1, 1, 2, 5, 10, 20, or 50 mm.

Fiber optic tapers (e.g. Edmund Optics part no. 55-139) are another kindof faceplate that can be used to separate the sample from solid-stateimage sensors or a thin-film array. Like a fiber faceplate, a fiberoptic taper (also called a fiber taper or simply a taper) comprises abundle of coherent optical fibers, but the fibers have larger diametersat one end than at the other end. As a result, the two functionalsurfaces of the taper have unequal areas, with the surface correspondingto the thicker ends of the fibers having greater area. In embodimentswhere solid-state image sensors are used, a sensor may be placedadjacent to the small surface of a fiber taper, so that the image of thesample (or a portion thereof) passing through the taper is magnified. Afixed individual sensor can thus acquire a sub-image corresponding to alarger area of the sample, without placing the sensor farther from thesample or losing resolution. Alternatively, a moveable sensor can bescanned over the small surface of the fiber taper, requiring smaller (orfewer) movements than would be necessary to image the same portion ofthe sample in the absence of the fiber taper (FIG. 4). In embodimentsemploying a thin film-array, the array can likewise be placed adjacentto the smaller surface of the fiber taper. This allows a thin-film arraysmaller than the sample to capture an image of the entire sample. It isalso possible to move a thin film array and a fiber taper in acoordinated fashion with respect to sample, such as by scanning over thesample, to permit imaging of very large samples.

In embodiments employing a thin-film array, certain sources of noiseassociated with dark signal discussed above can be mitigated by coolingthe photosensor array, since dark signal occurrence is often reducedwhen the sensor temperature is reduced. Cooling of the photosensor arraymay result in undesired cooling of the sample (the analyte array),however. In chemiluminescence applications, for example, cooling thesample may reduce the intensity of the light produced by the sample.Sample cooling can be mitigated by thermally isolating the photosensorarray from the sample by placing a thermally insulating material betweenthe sample and the array. Alternatively, the adverse effects of samplecooling can be avoided by timing of the measurement, i.e., by coolingthe sensor array before the sample is brought in close contact with thearray, then drawing the sample toward the array and conducting themeasurement quickly before the sample has time to cool. The same resultmay be achieved by heating the sample with an indium tin oxide (ITO)layer or by infrared radiation while cooling the sensor.

Descriptions of thin film arrays of the types described above and theiruse are found in Ovshinsky et al. (Energy Conversion Devices, Inc.) U.S.Pat. No. 4,788,594 (Nov. 29, 1988), Ovshinsky et al. (Energy ConversionDevices, Inc.) U.S. Pat. No. 4,853,785 (Aug. 1, 1989), Weisfield (XeroxCorporation) U.S. Pat. No. 5,619,033 (Apr. 8, 1997), Park et al. (dpiX,L.L.C.) U.S. Pat. No. 7,265,327 B1 (Sep. 4, 2007), Yuan et al. (DPIXLLC) U.S. Pat. No. 7,532,264 (May 12, 2009), Weisfield et al. (XeroxCorporation) U.S. Pat. No. 7,902,004 (Mar. 8, 2011), and Zentai et al.(Varian Medical Systems, Inc. and dpiX, L.L.C.) U.S. Pat. No. 8,232,531,B2 (Jul. 31, 2012).

The planar matrix that serves as a support for the array of molecularspecies can be either a slab gel in which electrophoresis has beenperformed to achieve the separation, a blotting membrane to which thebands or spots in a gel have been transferred, or any of a variety ofother spatial arrays or patterns that are obtained in a variety of waysand are used for a variety of purposes. Proteins, nucleic acids, orother biological species that have been electrophoretically separated ina slab gel are often transferred to a blotting membrane formed ofnitrocellulose, nylon, polyvinyl difluoride, or similar materials priorto identification and quantification. A common transfer technique iselectroblotting, in which flat surfaces of the gel and membrane areplaced in direct contact and an electric current is passed through boththe gel and the membrane in a transverse direction, thereby transferringthe species in a manner similar to that by which the species weremobilized within the gel. When the species are DNA fragments, thetransfer is termed a Southern blot after its originator, the Britishbiologist Edwin M. Southern. By analogy, the transfer of RNA fragmentsis termed northern blotting, and the transfer of proteins orpolypeptides is termed western blotting. Still further examples are“eastern” blots for post-translational modifications, and “far western”blots for protein interactions.

Electroblotting can be performed in either a wet, dry, or semi-dryformat. In wet blotting, the gel and membrane are layered over eachother in a stack which is immersed in a transfer buffer solution in atank on whose walls are mounted wire or plate electrodes. The electrodesare then energized to cause the solutes to migrate from the gel to themembrane. In semi-dry blotting, filter papers wetted with the transferbuffer solution are placed on the top and bottom of the stack with thegel and the membrane in between to form a “blotting sandwich.” Theelectrodes are then placed in direct contact with the exposed surfacesof the wetted filter papers. In dry electroblotting, no liquid buffersare used other than those residing in the gels. Descriptions of wet,dry, and semi-dry electroblotting and the associated materials andequipment are found in Margalit et al. (Invitrogen) United States PatentApplication Publications No. US 2006/0272946 A1 (Dec. 7, 2006), No. US2006/0278531 A1 (Dec. 14, 2006), and No. US 2009/0026079 A1 (Jan. 29,2009); Littlehales (American Bionetics) U.S. Pat. No. 4,840,714 (Jun.20, 1989); Dyson et al. (Amersham International) U.S. Pat. No. 4,889,606(Dec. 26, 1989); Schuette (Life Technologies, Inc.), U.S. Pat. No.5,013,420 (May 7, 1991); Chan et al. (Abbott Laboratories), U.S. Pat.No. 5,356,772 (Oct. 18, 1994); Camacho (Hoefer Scientific Instruments),U.S. Pat. No. 5,445,723 (Aug. 29, 2005); Boquet (Bertin & Cie), U.S.Pat. No. 5,482,613 (Jan. 9, 1996); and Chen (Wealtec Enterprise Co.,Ltd.) U.S. Pat. No. 6,592,734 (Jul. 15, 2003).

Regardless of the electroblotting format, the resulting electroblot isoften treated with detection reagents to render the bands or spots inthe blot detectable to the photosensors or photoresponsive elements inthe detector array by methods appropriate to the species in the bands orspots. In Southern and northern blots, for example, the detectionreagents are hybridization probes followed by a fluorescent orchromogenic dye. In western blots, the detection reagents are antibodiesfollowed by the use of conventional labeling techniques to detect theantibodies. Similar or analogous procedures, known among skilledbiochemists, are performed with far western blots and eastern blots.Treatments such as these can also be applied directly to gels.

Additional examples of matrices with two-dimensional arrays of molecularspecies are mass spectroscopy targets. Still further examples areproteins, nucleic acids, or other biological species that have beendeposited on a membrane or other support surface in regularly spaced orirregularly spaced two-dimensional arrays by such means aselectrospraying, vacuum deposition, and pin spotting.

In the claims appended hereto, the term “a” or “an” is intended to mean“one or more.” The term “comprise” and variations thereof such as“comprises” and “comprising,” when preceding the recitation of a step oran element, are intended to mean that the addition of further steps orelements is optional and not excluded. All patents, patent applications,and other published reference materials cited in this specification arehereby incorporated herein by reference in their entirety. Anydiscrepancy between any reference material cited herein or any prior artin general and an explicit teaching of this specification is intended tobe resolved in favor of the teaching in this specification. Thisincludes any discrepancy between an art-understood definition of a wordor phrase and a definition explicitly provided in this specification ofthe same word or phrase.

What is claimed is:
 1. A method of analyzing a plurality of analytesdetectable by light emission or absorption and arranged in atwo-dimensional analyte array supported by a planar matrix whose length,width, or both length and width measure a minimum of about 3 cm, themethod comprising: (a) placing the planar matrix having thetwo-dimensional analyte array supported thereon within 5 cm of adetector, wherein the detector comprises: a plurality of photoresponsiveelements arranged in a sensor array that is at least substantiallycoextensive with the planar matrix, thin-film addressing circuitry thatcontrols accumulation of energy by, and release of energy from, thephotoresponsive elements, and an imaging device configured to correlateenergy released from the photoresponsive elements in accordance withsites on the planar matrix and form a planar matrix image of the planarmatrix in full from the energy so released, wherein a transparentfaceplate is placed between the planar matrix and the detector, andwherein the transparent faceplate is deposited on or bonded directly tothe sensor array; (b) generating the planar matrix image in full usingthe detector in a manner that either compensates for or eliminates anyirregularities in light intensity across the planar matrix image thatare not representative of the two-dimensional analyte array; (c)generating a dark signal pattern for the sensor array and subtractingthe dark signal pattern from the planar matrix image, wherein generatingthe dark signal pattern comprises measuring a plurality of temperaturesat different locations within the sensor array to determine atemperature variation, and producing the dark signal pattern as afunction of the temperature variation; and (d) analyzing the planarmatrix image so generated.
 2. The method of claim 1, wherein thetransparent faceplate guides light from the planar matrix to theplurality of photoresponsive elements.
 3. The method of claim 1, whereinthe transparent faceplate is a fiber faceplate or a fiber taper, whereinthe transparent faceplate comprises light pipes, and wherein each of theplurality of photoresponsive elements receives light from a separategroup of light pipes.
 4. The method of claim 1, wherein a maximumthickness of the transparent faceplate is about 10 mm.
 5. The method ofclaim 1, wherein step (b) comprises applying flat field correction tocompensate for or eliminate the irregularities.
 6. The method of claim1, further comprising: cooling the sensor array to reduce dark signalnoise.
 7. The method of claim 6, wherein a thermally insulating materialis placed between the planar matrix and the sensor array.
 8. The methodof claim 6, wherein the cooling is stopped prior to step (a).
 9. Themethod of claim 1, wherein the photoresponsive elements are thin-filmfield effect transistors (TFT).
 10. The method of claim 1, wherein thephotoresponsive elements are complementary metal-oxide-semiconductors(CMOS).
 11. The method of claim 1, wherein the photoresponsive elementsare charge-coupled devices (CCD).
 12. The method of claim 1, wherein theplurality of analytes is detectable by detecting light absorption. 13.The method of claim 1, wherein the plurality of analytes is detectableby detecting light transmission.
 14. The method of claim 1, wherein theplurality of analytes is detectable by detecting light fluorescence. 15.The method of claim 1, wherein the plurality of analytes is detectableby detecting chemiluminescence or bioluminescence.